The Mechanism Of TWK-40 Regulating Rhythmic Defecation In C.elegans

Motor rhythm regulates various important physiological activities,such as walking gait,chewing,breathing,etc.In the digestive system,rhythmic defecation acts as an important way to directly regulate metabolites and is an indispensable part of life activities.Studying the regulation mechanism of defecation rhythm is of great significance for understanding the formation of biological rhythm and treating rhythm disorders(such as constipation).Caenorhabditis elegans(C.elegans)has a strict defecation cycle,and each cycle contains a precise step program(including anterior body wall muscle contraction a Boc,posterior body wall muscle contraction p Boc,expulsion Exp).Compared with mammals,defecation rhythm cycle of C.elegans is short and simple,but like mammals,this rhythm is regulated by precise intestinal and neural signals.In this paper,we use C.elegans as a model to study the cellular and molecular regulation mechanism of rhythmic defecation.In the paper,using the known defecation disorder mutants nlp-40(tm4085)and aex-2(sa3),the two-pore-domain potassium channel twk-40 with unknown function was screened,and it was found that the loss-of-function mutant twk-40(hp834,lf)can improve the Exp rhythm disorder of nlp-40 and aex-2 mutants,indicating that twk-40 regulates the C.elegans defecation rhythm.Surprisingly,twk-40(lf)itself did not exhibit abnormal Exp rhythm,but the prepared gain-of-function mutant twk-40(bln336,gf)caused serious Exp defects.Through fluorescence colocalization experiments,we observed that twk-40 is expressed in DVB,a key neuron that regulates C.elegans rhythm defecation.Expression of TWK-40(WT)in DVB neuron of twk-40(gf)mutant can partially rescue the Exp-deficient phenotype.Expression of TWK-40(bln336,GF)in DVB neuron of wild-type or twk-40(lf)mutant can imitate the Exp-deficient phenotype of twk-40(gf)mutant.The above experiments show that twk-40 may regulate worm DVB activity to control defecation rhythm.Subsequently,the calcium-sensitive protein GCa MP6.0s was expressed in DVB neuron and intestinal muscles that form synaptic function connection with DVB.We observed that the strong rhythmic calcium oscillation activity occurred synchronously with Exp.Further,we found that twk-40(gf)severely inhibited the calcium oscillation frequency and amplitude of DVB neuron.In contrast,twk-40(lf)caused the frequency of calcium oscillation in DVB neuron and intestinal muscles to increase,but did not change the amplitude of calcium oscillation.Neuron-specific rescue and overexpression experiments combined with real-time calcium imaging once again verified that twk-40 cellautonomously regulates the activity of DVB neuron,thereby regulating rhythmic defecation.Finally,we exogenously expressed twk-40 on HEK293 cell.Whole-cell recording found that TWK-40 produces an outward potassium channel current,which hyperpolarize the resting potential of cell membrane.The gain-of-function mutant TWK-40(bln336,GF)produces a stronger outward current and lower resting membrane potential.In short,this paper studies the cellular mechanism of twk-40 regulating C.elegans rhythmic defecation.The gain-of-function mutant of twk-40 will cause the two-pore-domain potassium channel current of DVB neuron to increase,so that the cell are hyperpolarized,inhibit the excitability of neuron,and further inhibit the excitability of intestinal muscles,eventually leading to the emergence of Exp defects phenotype in C.elegans.The results of this paper provide a basis for further research on rhythmic defecation,and at the same time provide a reference for the treatment of physiological diseases caused by abnormal defecation rhythms.